Gene transfer vectors require the efficient transduction of target cells, stable association with the host genome, and adequate transgene expression in the appropriate target cell, without associated toxic or immunological side effects. Currently available viral vector systems, including recombinant retroviruses, adenoviruses and adeno-associated viruses, are not suitable for efficient gene transfer into many cell types. Retroviral vectors require cell division for stable integration. Recombinant adenoviruses are not able to infect many cell types important for gene therapy, including hematopoietic stem cells, monocytes, T- and B-lymphocytes. Moreover, recombinant adeno-associated vectors (AAV) integrate with a low frequency.
First generation adenoviruses have a number of properties that make them an attractive vehicle for gene transfer (Hitt, M. M. et al. 1997 Advances in Pharmacology 40:137–205). These include the ability to produce purified virus at high titers in concert with highly efficient gene transfer of up to 8 kb long expression cassettes into a large variety of cell types in vivo, including non-dividing cells. Limitations of first generation adenoviruses include the development of immune responses to expressed viral proteins resulting in toxicity and virus clearance. The episomal status of adenoviral DNA within transduced cells is another limitation of first generation Ad vectors. Stable integration of adenovirus DNA into the host genome is reported only for wild-type forms of specific subtypes and appears not to occur in a detectable manner with E1/E3-deleted Ad 5 (adenovirus serotype 5) vectors widely used for gene transfer in vitro and in vivo [Hitt, M. M. et al. 1997 Advances in Pharmacology 40:137–205].
Recombinant AAV vectors (rAAV) integrate with a low frequency (about 1 out of 20,000 genomes) randomly as cocatemers into the host genome (Rutledge, E. A.; Russel, D. W. 1997 J. Virology, 71, 8429–8436). The presence of two AAV inverted terminal repeats (ITRs) and as yet unknown host cellular factors seem to be the only requirement for vector integration (Xiao, X., et al, 1997, J. Virology, 71, 941–948; Balague, C., et al. 1997, J. Virology, 71, 3299–3306; Yang, C. C. 1997, J. Virology, 71, 9231–9247). In the presence of the large AAV Rep proteins, AAV integrates preferentially into a specific site at human chromosome 19, called AAVS1 (Berns, K. I., 1996, Fields Virology, Fields, B. N. et al. (ed) Vol. 2, Lippincott-Raven, Philadelphia, Pa., 2173–2220). The AAV capsid is formed by three coat proteins (VP1–3), which interact with specific heparin sulfates on the cell surface and probably with specific receptor(s). However, many cell types, including hematopoietic stem cells, lack these structures so that rAAV vectors based on AAV2 cannot infect or transduce these cells (Malik P. et al., 1997, J. Virology, 71, 1776–1783; Quing, K. Y., et al. 1998, J. Virology, 72, 1593–1599). Other disadvantages of rAAV vectors include the limited insert size (4.5–5 kb) that can be accommodated in rAAV vectors lacking all viral genes and low transducing titers of rAAV preparations.
Adenovirus infection is initiated by attaching to the cell surface of Ad 5 via its fiber protein (for a review, see Shenk, T. 1996 Fields Virology, Vol. 2, Fields, B. N. et al. (ed) Vol. 2, Lippincott-Raven, Philadelphia, Pa., 2111–2148). The distal, C-terminal domain of the trimeric fiber molecule terminates in a knob, which binds to a specific cellular receptor identified recently as the coxackie-adenovirus receptor (CAR) (Bergelson, J. M. et al. Science, 275, 1320–1323). After binding, in an event independent of virus attachment, Arg-Gly-Asp (RGD) motifs in the penton base interact with cellular integrins of the α3 and β5 types. This interaction triggers cellular internalization whereby the virion achieves localization within the endosome. The endosomal membrane is lysed in a process mediated by the penton base, releasing the contents of the endosome to the cytoplasm. During these processes, the virion is gradually uncoated and the adenoviral DNA is transported to the nucleus where replication takes place. The terminal protein, which is covalently attached to the viral genome and the core protein V that is localized on the surface of the cores have nuclear localization signals (NLSs) (van der Vliet, B. 1995, The Molecular Repertoir of Adenoviruses, Vol. 2, Doerfler, W. and Boehm, P. (ed.), Springer Verlag, Berlin, 1–31). These NLSs play a crucial role in directing the adenoviral genome to the nucleus and probably represent the structural elements which allow adenovirus to transduce non-dividing cells. When the double-stranded, linear DNA reaches the nucleus, it binds to the nuclear matrix through its terminal protein.
Since the cell types that can be infected with Ad5 or Ad2 vectors are restricted by the presence of CAR and specific integrins, attempts have been made to widen the tropism of Ad vectors. Genetic modification of adenovirus coat proteins to target novel cell surface receptors have been reported for the fiber (Krasnykh, V. et al. 1998 J. Virology, 72, 1844–1852, Krasnykh, V. et al. 1996 J. Virology, 70, 6839–6846, Stevenson, S. D., et al. 1997, J. Virology, 71, 4782–4790), penton base (Wickham, T. J., et al. 1996, J. Virology, 70, 6831–6838; Wickham, T. J., et al. 1995, Gene Therapy, 69, 750–756), and hexon proteins (Crompton, J., et al. 1994, J. Gen. Virol. 75, 133–139). The most promising modification seems to be the functional modification of the fiber protein or more specifically of the fiber knob as the moiety, which mediates the primary attachment. Two groups have reported the generation of fibers consisting of the Ad5 tail/shaft and the knob domain of Ad3 (Krasnykh, V. et al. 1996 supra, Stevenson, S. D., et al. 1997, supra). Recently, recombinant adenoviruses with fibers containing C-terminal poly-lysine, gastrin-releasing peptide, somatostatin, E-selectin-binding peptide, or oligo-Histidines were produced in order to change the native tropism of Ad5. Krasnikh et al. found (Krasnykh, V. et al. 1998 supra) that heterologous peptide ligands could be inserted into the H1 loop of the fiber knob domain without affecting the biological function of the fiber. Based on studies with other Ad serotypes, it appears that the length of the fiber shaft is a critical element, determining the efficiency of interaction with cell surface integrins and the internalization process. Thus far, there is no reported data demonstrating successful retargeting of Ad5 vectors for a specific cell type.
Therefore, there is a present need for an improved adenovirus vector which can be targeted efficiently to a variety of cell types and tissues and remain stably integrated in the host genome with minimal antigenicity to the host. The present invention discloses novel chimeric adenoviral (Ad) Ad-AAV vectors, which express a modified fiber protein on their capsid, for specifically targeting the vector. Methods of making, uses and advantages of these vectors are described. In addition, the alteration described for the knob and shaft domains of the fiber protein provide a novel approach to retarget any adenovirus serotype for cell specific infection.